Carboxylated heterocyclic compounds and methods of synthesis

ABSTRACT

Compositions of the present invention comprise carboxylated heterocyclic compounds, including carboxyflosequinan. The methods of the present invention also comprise the synthesis of carboxyflosequinan.

FIELD OF THE INVENTION

[0001] The present invention teaches compositions comprisingcarboxylated heterocyclic compounds and the synthesis of the same.

BACKGROUND

[0002] A variety of heterocyclic compounds have been described as havingvarious pharmaceutical applications. However, the synthesis of suchcompounds, especially on a large scale, is often labor-intensive,expensive and time consuming. What is needed therefore, is a simplifiedand economical method for the synthesis and purification of heterocycliccompounds.

SUMMARY OF THE INVENTION

[0003] The present invention relates to compositions comprisingcarboxyflosequinan and the synthesis of the same.

[0004] In one embodiment, the present invention teaches a carboxylatedheterocyclic compound corresponding to3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone(carboxyflosequinan) and derivatives thereof.

[0005] In one embodiment, the present invention teaches providing,3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone and a first acidfollowed by the reaction of said3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone and first acid underconditions such that 3-carboxymethylthio-7-fluoro-1-methyl-4-quinoloneis produced.

[0006] In another embodiment the present invention further contemplatesthe reaction of 3-carboxymethylthio-7-fluoro-1-methyl-4-quinolone with asecond acid and a peroxide under conditions such that3-Carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone is produced.

DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1 shows the chemical structure of3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone.

[0008]FIG. 2 displays a scheme for the synthesis of3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone.

[0009]FIG. 3 depicts the results of enzyme (PKC) inhibition assays with3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone.

DEFINITIONS

[0010] As used herein carboxyflosequinan refers to the chemicalcomposition designated as3-carboxymethylsufinyl-7-fluoro-3-methyl-4-quinolone having the chemicalstructure corresponding to:

[0011] As used herein, the phrase “flosequinan” and a “a racemic mixtureof flosequinan” refers to7-fluoro-1-methyl-3-(methylsulphinyl)-4(1H)-quinolinone which may alsobe described as 7-fluoro-1-methyl-3-(methylsulfinyl)-4(1H)-quinolone)and as 7-fluoro-1-methyl-3-methylsulfinyl-4-quinolone having thechemical structure of:

[0012] As used herein, “room temperature”, “RT” or “ambient temperature”is approximately 18° C. to 25° C.

[0013] As used herein, “overnight” is approximately 8 hours, morepreferably 12 hours, more typically 17 hours, but can be up toapproximately 30 hours.

[0014] As used herein, a “heterocyclic” compound refers to a compoundcomprising a ring composed of atoms of more than one kind.

[0015] As used herein, a “catalyst” refers to a substance that, whenadded to a reaction mixture, changes (e.g. speeds up) the rate ofattainment of equilibrium in the system without itself undergoing apermanent chemical change. Examples of suitable catalysts contemplatedfor use in the present invention include, but are not limited to,antimony chloride and carbon tetrabromide.

[0016] As used herein, a “solvent” refers to a substance that willdissolve other substances. An “organic solvent” is an organic substancethat will dissolve other substances. Examples of solvents suitable foruse in embodiments of the present invention include, but are not limitedto dichloromethane and acetonitrile.

DETAILED DESCRIPTION OF THE INVENTION

[0017] In one embodiment, the present invention describes a compositioncomprising carboxyflosequinan. In another embodiment, the presentinvention teaches methods for the synthesis of carboxyflosequinan.

[0018] The present invention also contemplates the formulation ofcarboxyflosequinan as a pharmaceutically acceptable salt. In addition,pharmaceutical formulations of carboxyflosequinan may also containbinders, fillers, carriers, preservatives, stabilizing agents,emulsifiers, buffers and excipients as, for example, pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharin, cellulose, and magnesium carbonate. The present inventionalso contemplates the administration of carboxyflosequinan as apharmaceutically acceptable salt or formulation. The present inventionalso contemplates the administration of carboxyflosequinan andcarboxyflosequinan formulations to a subject.

[0019] Methods of producing a racemic mixture of flosequinan, as set outin U.S. Pat. Nos. 5,079,264 and 5,011,931 to MacLean et al., are herebyincorporated by reference. In one embodiment, flosequinan is preparedaccording to the protocol set out in Example 2.

[0020] Without limiting the invention to any particular mechanism,carboxyflosequinan is an enzyme inhibitors. In a specific example,carboxyflosequinan inhibit protein kinase C (herein after PKC). Thiseffect of carboxyflosequinan on enzyme activity, more particularly onPKC, has utility in therapeutics.

EXPERIMENTAL

[0021] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0022] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); L (liters); ml (milliliters); °C. (degrees Centigrade).

[0023] All bracketed numbers [e.g. “(1)”] after the chemical name of acompound, refer to the corresponding chemical structure as designated bythe same bracketed number in FIG. 1.

[0024] All NMR spectra were recorded using Varian-Gemini 300 MHzSpectrometer.

[0025] In Examples 1, unless otherwise stated, the source for thechemical reagents was Aldrich, Milwaukee, Wis., U.S.A. (unless a reagentwas synthesized de novo, as described in the examples). Flosequinan wassynthesized according to the protocol provided in Example 2, unlessspecified otherwise.

EXAMPLE 1 Synthesis of3-Carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone (5).

[0026] To an efficiently stirred and gently cooled with dry-ice acetonemixture of 12 ml of thionyl chloride (SOCl₂) and 3 ml of pyridine (Py)at −3°° C. was added flosequinan (3.59 g, 15 mmol) (1) in a few portionsover a period of approximately 1 min. During that time cooling wasapplied to keep the temperature in the range 0-6° C. The mixture wasstirred at 0° C. for 5 min, cooled to −5° C. and poured as a thin streaminto 350 ml of ice-water with efficient stirring. After 10 min stirringat 0° C. a solid was filtered off, washed with water, and dried overphosphorus pentoxide under high vacuum. Yield 2.82 g (74%) of a crudeproduct that was ˜95% pure by ¹H NMR. The crude product(3-Chloromethylthio-7-fluoro-1-methyl-4-quinolone) (2) was used in thenext step without further purification.

[0027] To efficiently stirred suspension of sodium cyanide (490 mg, 10mmol) in dry DMSO (15 ml) at room temperature under a N₂ atmosphere wasadded crude 3-chloromethylthio-7-fluoro-1-methyl-4-quinolone (1.031 g, 4mmol) (2) in a few portions. The mixture was stirred for 1 h and pouredinto diluted H₂SO₄ with ice. The solid was filtered off. The filtratewas extracted twice with ethyl acetate, the combined extracts were driedover anhydrous Na2SO₄ and concentrated. The residue was combined withthe solid and was chromatographed on silica gel with hexane-ethylacetate (gradient 2:1,1:1,1:2) to give 644 mg (65%) of the product as abrownish solid. The product(3-Cyanomethylthio-7-fluoro-1-methyl-4-quinolone; 575 mg) (3) wasfurther purified by recrystallization from methanol to give 382 mg ofbrownish crystals.

[0028] A mixture of 3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone (265mg, 1.067 mmol) (3) and 3N hydrochloric acid (8 ml) was refluxed under aN₂ atmosphere for 2.5 h. The hot mixture was diluted with water (1 ml )and allowed to cool to room temperature. A solid that precipitated wasfiltered off and dried under high vacuum. The yield of3-Carboxymethylthio-7-fluoro-1-methyl-4-quinolone (4) was 270 mg(94.7%).

[0029] 50% hydrogen peroxide (57ml, 33.6 mg, 0.988 mmol) was added to asolution of 3-carboxymethylthio-7-fluoro-1-methyl-4-quinolone (4) inacetic acid (3.6 ml) at 60° C. and the mixture was stirred at 55° C. for4 h. The hot mixture was diluted with water (12ml) and cooled to 0° C. Awhite solid that precipitated was filtered off and dried under highvacuum. The yield of3-Carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone was 185 mg (72.7%)(5).

EXAMPLE 2

[0030] In this example flosequinan is prepared according to thefollowing protocol:

[0031] A. Preparation of flosequinan

[0032] i. Step I

[0033] In a clean and dry 12 L glass reactor equipped with a backsuction trap plus a NaOH (25%) trap at the outlet and a back suctiontrap in the inlet, 3.840 L of toluene were charged and cooled to −45° C.using a dry ice-acetone bath. Using appropriate safety precautions, 832g of phosgene were then passed through the cold toluene while stirringto prepare a 20% (wt/wt) solution. The addition of the phosgene tookapproximately 3.5 hours.

[0034] Separately, into a clean and dry 22 L glass reactor equipped withthe above-described types of back suction traps, 399 g of startingmaterial (formula I):

[0035] was added with stirring to 4.37 L of deionized water. A separate6.8% solution of sodium carbonate in water was also prepared by adding297 g of sodium carbonate to 4.37 L of deionized water. Using a cleanaddition funnel, the sodium carbonate solution was then slowly addedwith stirring to the suspension of the starting material, to create abrown-colored solution.

[0036] In preparation for the reaction step, the phosgene solution waswarmed from −45° C. to −15° C. and the mixture of the starting materialand the sodium carbonate was cooled to 10° C. The phosgene solution wasthen added over approximately 1.5 hours with stirring to the brownsolution. The reaction mixture was stirred overnight allowing thedesired intermediate-A (formula II):

[0037] to precipitate out. A sample was removed for NMR assessment andthe precipitate was filtered on a 4 L sintered glass funnel. Thefiltrate was washed with 2×500 ml aliquots of cold deionized water anddried under a vacuum at approximately 50° C. for 16 hours.

[0038] A 93.4% lot yield of 435 g of intermediate-A (formula II) wasobtained. This procedure was repeated three more times, starting withapproximately 400 g of starting material each time. Lot yields of 448 g(94.5%), 449 g (95.9%), and 459 g (96.8%) were obtained.

[0039] ii. Step II

[0040] In a 22 L oven dried glass reactor equipped with a reflexcondenser, addition funnel and temperature recorder, 11.40 L ofanhydrous tetrahydrofuran (THF) were added under nitrogen. To thisreactor were also added 409 g of 60% sodium hydride in oil. Eightapproximately equal portions of intermediate A (formula II) were thenadded to the reactor, totaling 883 g altogether. As this reaction isexothermic, care was taken to avoid excessive heat and bubbling. Finaltemperature was 40° C., with a maximum observed temperature of 41° C.The reaction mixture was stirred until hydrogen gas evolution ceased.

[0041] To the reaction mixture was then slowly added 575 ml (766.4 g) ofdimethyl sulfate, keeping the temperature below 50° C. Upon completion,the reaction mixture was stirred at 50° C. for 3 hours with the refluxcondenser on. A sample was removed for NMR assessment, and the heat wasturned off before stirring overnight.

[0042] In the morning, the stirring was stopped and the clear liquid ontop was siphoned off. This liquid was filtered using a 2-3 inch thickCelite pad in a 2 L sintered glass funnel. The residue cake was keptcovered to minimize contact with atmospheric moisture. The residue wascollected and washed with 4 aliquots of anhydrous THF. The filtrate andthe washings were evaporated to dryness using a rotary evaporator andthe residue obtained was dried under vacuum at approximately 36-38° C.overnight. A sample was removed for NMR assessment of the amount ofunreacted dimethyl sulfate present. The dried residue was then added to1600 ml of a 1:3 toluene:hexane mixture and vigorously stirred. Thismixture was then filtered and washed with 2×700 ml washings of 1:3toluene:hexane mixture. A reference sample was removed for NMRassessment and the residue was dried at 51-50° C. under vacuum for 36hours.

[0043] This batch yielded 871 g of intermediate-B (formula III):

[0044] for a lot yield of 91.6%. Another 907.1 g of intermediate-A wassubjected to the procedure of step II, in which the amounts of reactantsand solvents was proportionately adjusted with a yield of 850 g (87%).

[0045] iii. Step III

[0046] In an oven dried 12 L glass reactor equipped with a stirrer,temperature recorder and addition funnel, 2550 ml of anhydrous toluenewas added under nitrogen. Then 236 g of 60% sodium hydride in oil wasadded, all at room temperature. The reaction mixture was heated withcontinuous stirring to 75° C. using a heating mantel. Then 1.59 L ofanhydrous dimethyl sulfoxide (DMSO) were added slowly and carefully over45 minutes taking care to avoid excessive bubbling. The reaction mixturewas stirred for one hour at 70-72° C. until clear and hydrogen gasevolution ceased. The heating mantel was turned off and a water bath wasused to cool the reaction mixture to 30° C.

[0047] To this mixture, 538.2 g of dry intermediate-B (formula III) wasadded slowly in portions, keeping the temperature no higher than 35° C.Then 1.9 L of anhydrous DMSO was added, again keeping the temperature nohigher than 35° C. The reaction mixture was stirred under nitrogen forone hour, allowing the mixture to cool to 26°. The reaction mixture wasthen quenched slowly and carefully with 320 ml of methanol. Theresulting suspension was then added slowly and with vigorous stirring toa 22 L reaction vessel containing 12.760 L of diethyl ether.

[0048] After stirring was stopped, the upper ether layer was siphonedoff and the brown oil lower layer was washed with 520 ml of fresh ether.The oily yellow residue was triturated with 2600 ml of deionized wateruntil a yellow precipitate formed. This precipitate was filtered using a2 L sintered glass funnel and the solid residue was washed with threealiquots of 130 ml cold deionized water. A reference sample was taken toassess the residue. The residue was dried under vacuum at 50-53° C. for23 hours.

[0049] This procedure produced 243 g of intermediate C (formula IV):

[0050] which represents a 38.4% yield. Two other batches ofintermediate-B were treated according to this Step III procedure, withproportionate adjustments to the amounts of reactants and solvents. Thefirst additional batch of 538.2 g intermediate-B produced a 192 g(30.4%) yield, and the second additional batch of 87.38 g ofintermediate-B produced a yield of 42 g (40.9%).

[0051] iv. Step IV

[0052] In a 12 L oven dry glass reactor equipped with a stirrer,temperature recorder and addition funnel which has been dried bynitrogen flow for 30 minutes the following chemicals were charged: 7.990L of triethyl orthoformate; 696 g of intermediate-C; 324 ml ofpiperdine; and 296 ml of acetic acid. The reaction mixture was heatedunder nitrogen to reflux at approximately 105° C. for 2 hours. A samplewas removed to assess the progress of the reaction step by NMR.

[0053] Using a water bath, the reaction mixture was then cooled to roomtemperature and stirred for 30 minutes. The final product precipitatedout and was collected by filtration on a 4 L sintered glass funnel. Theresidue was washed with 3×700 ml aliquots of diethyl ether, and a samplewas removed for NMR assessment. The residue was dried under vacuum at50-51° C. for 17 hours. A sample of the dried flosequinan product(formula V):

[0054] was removed for NMR assessment. 547 g (75.3%) yield offlosequinan was obtained (an additional 47 g of product was scraped fromthe bottom of the sintered glass filter but was not included in thistotal yield calculation).

EXAMPLE 3

[0055] In this example, carboxyflosequinan was subjected to biochemicalenzyme assays and radioligand binding assays to determine its percentinhibition of a variety of enzyme activities. Tamaoki and Nakano “Potentand specific inhibitors of protein kinase C of microbial origin”Biotechnology 8:732 (1990); Wilkinson et al. “Isoenzyme specificity ofbisindolymaleimides, selective inhibitors of protein kinase C” Biochem.J. 294:335 (1993); Tamaki el al. “Staurosporine, a potent inhibitor ofphospholipid/Ca++ dependent protein kinase” Biochem. Biophys. Res. Comm,135:397 (1986). A brief summary of the conditions for each assay isprovided below:

[0056] Protein Serine/Threonine Kinase PKCα: Human recombinant enzymefrom Sf9 insect cells was used in the assay. The substrate was 200 μg/mlhistone. The reaction was incubated 10 mins at 25° C. in 20 mM Hepes, 10mM MgCl₂, 0.1 mM CaCl₂. [³²P]histone was quantitated.

[0057] Protein Serine/Threonine Kinase PKC, non-selective: The enzymewas obtained from rat brain and the substrate was 370 μg/ml histone. Thereaction was pre-incubated 5 min at 25° C., followed by a 15 minincubation at 25° C. in a buffer of 20 mM Tris-HCl, 10 mM MgCl₂.H_(o)and 0.1 mM CaCl₂.2H_(o), pH 7.4. [³²P]histone was quantitated.

[0058]FIG. 3 projects data for carboxyflosequinan in the assaysdescribed above. In these protein kinase assays, carboxyflosequinan wasused in varying concentrations (in 1% DMSO as the vehicle). For theprotein serine/threonine kinase (PKC, non-selective), carboxyflosequinanwas tested at a concentrations of 100 μM, 300 μM, and 1000 μM.Significant (e.g., greater than 50%) inhibition was observed at allthree concentrations of carboxyflosequinan. (See, FIG. 3).

[0059] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described invention will be apparent to those skilledin the art without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in the art are intendedto be within the scope of the following claims.

1. A composition comprising3-carboxymethylsulfinyl-7-fluoro-3-methyl-4-quinolone having thecorresponding structure:


2. A method, comprising: a) providing: i)3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone; and ii) a first acid;b) reacting said 3-cyanomethylthio-7-fluoro-1-methyl-4-quinolone andsaid first acid under conditions such that3-carboxymethylthio-7-fluoro-1-methyl-4-quinolone is produced.
 3. Themethod of claim 2 further comprising: c) reacting said3-carboxymethylthio-7-fluoro-1-methyl-4-quinolone with; i) a second acidand ii) a peroxide under conditions such that3-carboxymethylsulfinyl-7-fluoro-1-methyl-4-quinolone is produced.